Enzolution® MDA5 ATPase Assay System

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Enzolution MDA5 ATPase Assay System Technical Manual

A comprehensive assay system for measuring the activity of MDA5 ATPase Activity.

Includes enzyme, substrate, plates, and buffers
For use with the trusted Transcreener ADP² Assay (Sold Separately)
Easy-to-use and HTS-ready
Screen & perform dose-response assays quickly in your lab

Is your plate reader compatible? Find out now!

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SKU: N/A Category: Enzyme Assay Systems

Enzolution MDA5 ATPase Assay System

Name: Enzolution® MDA5 ATPase Assay System
SKU: 21396
Availability: InStock

The Enzolution MDA5 Assay System provides researchers with an assay tool that will simplify and advance their exploration of novel innate immunity modulators. The assay is designed to screen and profile diverse compound libraries in a high-throughput format to identify small molecule inhibitors.

The Enzolution Assay System is intended for use with the trusted Transcreener ADP² Assay (sold separately). The Transcreener ADP² Assay is the only commercially available ADP assay that directly detects ADP production, i.e. without the use of coupling enzymes. The direct detection format minimizes compound interference, producing trustworthy results with Z’ values greater than 0.7. The assay is an extensively validated ADP detection method with use in over millions of wells by pharma.

When purchasing the Enzolution Assay System, it is important to choose the assay system readout that corresponds to the FP, FI, or TR-FRET configuration of your Transcreener ADP² Assay.

A Complete Assay Solution for MDA5 Discovery

Enzolution MDA5 ATPase Assay System Schematic

Need Protocol Help? Download Technical Manual Here

Poster: Interrogating RIG-I and MDA5 with the Transcreener ADP²Assay

Specifications

What’s Included

Enzolution MDA5 FP & FI Assay System
	1K		10K
Component	Part#	Qty	Part#	Qty
Recombinant MDA5, Active, Human	2298	10 µg	2299	100 µg
Poly(I:C) dsRNA	2301	24 µL	2302	240 µL
MnCl2, 1M	2303	30 µL	2304	300 µL
Enzyme Assay Buffer C, 10X	2292	2 mL	2295	20 mL
DTT, 1M	2296	100 µL	2297	1 mL
Corning 384-Well Black Assay Plates (#4514)	2229	3-Pack (1,000+ Assays)	2230	30-Pack (10,000+ Assays)

Enzolution MDA5 TR-FRET Assay System
	1K		10K
Component	Part#	Qty	Part#	Qty
Recombinant MDA5, Active, Human	2298	10 µg	2299	100 µg
Poly(I:C) dsRNA	2301	24 µL	2302	240 µL
MnCl₂, 1M	2303	30 µL	2304	300 µL
Enzyme Assay Buffer C, 10X	2292	2 mL	2295	20 mL
DTT, 1M	2296	100 µL	2297	1 mL
Corning 384-Well White Assay Plates (#4513)	2231	3-Pack (1,000+ Assays)	2232	30-Pack (10,000+ Assays)

What You Will Need

Component	Notes
Transcreener ADP Assay	Available for separate purchase through BellBrook Labs. View Transcreener ADP Assay to learn more. Choose the readout configuration (FP, FI, or TR-FRET) that is the best fit based on preference and plate reader compatibility.
Ultrapure Nuclease Free Water	Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem.
Plate Reader	A multi-detection microplate reader configured to measure output of the ADP tracer is required.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense submicroliter volumes into 384-well plates.

A Simple Two-Step Protocol

The MDA5 reaction begins upon addition of RNA/ATP substrate mixture. The reaction occurs for 120 minutes at 30°. ADP detection reagents are added (Transcreener ADP² Antibody & Tracer) and incubated for 60 minutes at room temperature. Read the plate on a compatible reader. That’s it!

Robust HTS-Ready Assay

Data confirming the robustness of the MDA5 assay. A robust system like this, is necessary to ensure the precision and efficiency required to scale-up assays for high-throughput screening. The Z’ shown here (Z’ = 0.83), demonstrates the assay’s suitability for HTS.

MDA5 Assay Shows Assay Robustness for HTS

Direct MDA5 Detection with the Trusted Transcreener ADP² Assay

Transcreener assays have fewer reagents and a less complex mechanism than any other nucleotide detection assay, which generally require coupling enzymes to convert a nucleotide to a product that can generate a signal with a reporter enzyme. The direct detection Transcreener format decreases the risk of false positives or missing a hit.

BellBrook’s Recombinant MDA5 Enzyme (Included in Assay System)

Active, human MDA5 (AA 299-1025) protein expressed and purified from Baculovirus. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >90% in a composition of 20 mM HEPES, 150 mM NaCl, 10% glycerol, and 2 mM TCEP (pH 7.5). Currently used with the Transcreener assay at a concentration of 3.47 nM in a 10 μL reaction.

Explore the MDA5 Enzyme

DNA Damage Response and Innate Immunity Preview

Download the free eBook, The DNA Damage Response and Innate Immunity, to explore MDA5 & Targets Related to the Innate Immune Response.

MDA5 Assay Validation Under Initial Velocity Conditions

Raw polarization signal (mP) is converted to ADP formed using a a standard curve. Under initial velocity conditions, we recommend selecting an enzyme concentration that produces a 50-80% change in signal, corresponding to the linear phase of the reaction after conversion of values to ADP formation. Here, we use 100 µM ATP with 10 ng/µL dsRNA substrate. The enzyme buffer included 50 mM Tris, 2.5 mM MgCl₂, 0.01% Brij-35, 1.25 mM MnCl₂, and 5 mM DTT. The enzyme reaction took place for 120 minutes at 30°C. The ADP Detection Mix included 1X Stop & Detect Buffer B, 4 nM ADP² Alexa Fluor 633 Tracer, and 82 µg/mL ADP antibody. The Detection Mix was added and incubated for 60 minutes at room temperature.

Enzyme Titration – FP Readout

MDA5 Titration New Enzyme With FP Readout

Linear Response

Using the MDA5 Assay to Discover Novel Innate Immunity Modulators

1280 compounds were screened from the Tocris 2.0 Library set. An interference screen was performed to eliminate compounds interfering with detection reagents. A total of 14 potential inhibitors were identified with polarization values ≥ 3 standard deviations above the mean, in which 10 showed no interference with the assay. A selected hit from the pilot screen (CBA) and the control compound were tested in dose-response mode.

Pilot Screen of 1280 Small Molecules

Dose Response Mode

IC₅₀ (CBA) = 4.97 μM
IC₅₀ (Suramin) = 0.341 μM

Choose the Fluorescent Detection Mode That Best Suits Your Preference

Choose the readout configuration that is the best fit for your lab based on preference and plate reader compatibility. The assay is available with an FP, FI, or TR-FRET readout. Reference our Instrument Compatibility resource to determine the detection mode compatible with your plate reader.

Fluorescent Intensity

TR-FRET

Let Us Do The Work: MDA5 Assay Services

Interested in moving your MDA5 program forward, but don’t want the hassle of bringing an assay in-house? BellBrook’s expert scientists have you covered. Our team of biochemical and enzymology expert scientists will work diligently to progress your lead candidates through the early-stages of drug discovery. The best part – we are a small custom shop. We can customize assay conditions to suit your project needs, produce results faster, and you will directly collaborate with one of our expert scientists.

Lead Discovery Services Include:

Inhibitor Screening – To identify or confirm activity with the target.
Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC₅₀ results.
Residence Time Measurements – Determination of k_off using ‘jump dilution’ enzymatic assay method.
Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.
Triaging Non-stoichiometric Inhibitors – Run assays under different conditions such as varying detergent and enzyme concentrations.
Evaluate Compound-Target Binding – Run thermal shift assays to confirm compound-target engagement via shifts in melting temperature.

Contact a Scientist About Services Today

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

Interrogating RIG-I and MDA5 with the Transcreener ADP² Assay

RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated gene 5) are members of the RLR (RIG-I-like receptors) family of immune sensors that bind double-stranded RNA (dsRNA) and trigger an IFN1 response in response to microbial infection and tumorigenesis. RLR agonists are being investigated for enhancing tumor immunity, and related RNA helicases in the DEAD box (DDX) helicase family are being targeted for cancer; therefore robust HTS assays for RIG-I and MDA5 are needed. RNA helicases use cycles of ATP binding and hydrolysis to drive local unwinding of dsRNA, so activity can be measured by detecting ADP formation.

Here, we:

Use the Transcreener ADP² Assay to develop a high throughput assay for RIG-I and MDA5 with FP, FI, or TR-FRET readouts
Produced recombinant RIG-I and MDA5 in baculovirus-infected insect cells and determined their RNA and ATP requirements
Optimized assays for initial velocity detection of RNA-dependent ATPase activity with Z’ values > 0.7
Validated the assays for HTS by screening a collection of bioactives (Tocris 2.0) and confirming the hits in dose response assays

Articles

Resources

Find the right resources to help you get to your solution faster.

FAQ

Protocols

Publications

Instrument Compatibility

Application Notes

Posters

Contact Us to Learn More

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

“Some CROs reserve their best teams for high profile customers. If you’re not right there with them you get less experienced teams… BellBrook understands enzyme kinetics, so it made that part of the project so much easier. The larger CRO didn’t seem to have the expertise we needed in this area.”

- BioPharma Lead Discovery Program Manager

“The ADP assay is a great choice for measuring activity for any ADP-generating enzyme in both real time and endpoint formats. The universal detection system has been very useful for more challenging targets.”

- Pharmaceutical Research Scientist

“Excellent blend of simplicity & good science. Excellent technical support”

- Director Pharma Drug Discovery Center

“The assay itself has good reproducibility and provides a clear readout on our enzyme activity. The most important piece, however, is the level of attention to our project and rapid turnaround time. The staff at BellBrook knows what they are doing.”

- Biotech Program Manager

“The Transcreener AMP/GMP Assay is extremely straightforward to use and is very sensitive. In a UV-based enzyme assay I needed 1 µM enzyme to see a decent signal; however using the Transcreener AMP assay I only need 10 nM. Also, BellBrook Labs have been very helpful with the assay development. Their representative has been very willing to quickly help me get my assay up and running.”

- University Research Scientist

“Through the process of developing and validating an assay for HTS applications, I generated a very nice data package of robust high quality data.”

- Biotech Research Scientist

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BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400

info@bellbrooklabs.com

Transcreener ADP² Kinase Assay Kits – FP, 1,000 Assay, 384 Well

Enzolution MDA5 ATPase Assay System

A Complete Assay Solution for MDA5 Discovery

Poster: Interrogating RIG-I and MDA5 with the Transcreener ADP²Assay